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Metabarcoding studies of fungal communities rely on curated databases for assigning taxonomy. Any host or other nonfungal environmental sequences that are amplified during polymerase chain reaction (PCR) are inherently assigned taxonomy by these same databases, possibly leading to ambiguous nonfungal amplicons being assigned to fungal taxa. Here, we investigated the effects of including nonfungal outgroups in a fungal taxonomic database to aid in detecting and removing these nontarget amplicons. We processed 15 publicly available fungal metabarcode data sets and discovered that roughly 40% of the reads from these studies were not fungal, although they were assigned as Fungus sp. when using a database without nonfungal outgroups. We discuss implications for metabarcoding studies and recommend assigning taxonomy using a database with outgroups to better detect these nonfungal amplicons.

This version of the article has been accepted for publication, after peer review and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at https://doi.org/10.1007/s13225-021-00493-7. Fungi are highly important biotic components of terrestrial ecosystems, but we still have a very limited understanding about their diversity and distribution. This data article releases a global soil fungal dataset of the Global Soil Mycobiome consortium (GSMc) to boost further research in fungal diversity, biogeography and macroecology. The dataset comprises 722,682 fungal operational taxonomic units (OTUs) derived from PacBio sequencing of full-length ITS and 18S-V9 variable regions from 3200 plots in 108 countries on all continents. The plots are supplied with geographical and edaphic metadata. The OTUs are taxonomically and functionally assigned to guilds and other functional groups. The entire dataset has been corrected by excluding chimeras, index-switch artefacts and potential contamination. The dataset is more inclusive in terms of geographical breadth and phylogenetic diversity of fungi than previously published data. The GSMc dataset is available over the PlutoF repository.

Meta-amplicon studies of fungal communities rely on curated databases for assigning taxonomy. Any host or other non-fungal environmental sequences that are amplified during PCR are inherently assigned taxonomy by these same databases, possibly leading to ambiguous non-fungal amplicons being assigned to fungal taxa. Here, we investigated the effects of including non-fungal outgroups in a fungal taxonomic database to aid in detecting and removing these non-target amplicons. We processed 15 publicly available fungal meta-amplicon data sets and discovered that roughly 40% of the reads from these studies were not fungal, though they were assigned asFungus sp. when using a database without non-fungal outgroups. We discuss implications for meta-amplicon studies and recommend assigning taxonomy using a database with outgroups to better detect these non-fungal amplicons.

Microbes have fundamental roles underpinning the functioning of our planet, they are involved in global carbon and nutrient cycling, and support the existence of multicellular life. The mangrove ecosystem is nutrient limited and without microbial cycling, life in this harsh environment would likely not exist. The mangroves of Southeast Asia are the oldest and most biodiverse of all the planets. They have vital roles helping to prevent shoreline erosion, act as nursery grounds for many marine species and contain significant stocks of sequestered carbon. Despite these recognised benefits and the importance of microbes in these ecosystems, studies examining the mangrove microbiome are scarce, especially in the Southeast Asian biodiversity hotspot. Here we examine the microbiome ofAvicenia albaandSonneratia albaand identify a core microbiome of 81 taxa, a further eight taxa (Pleurocapsa, Tunicatimonas, Halomonas, Marinomonas, Rubrivirga, Altererythrobacte, Lewinella, andErythrobacter) were found to be differentially abundant suggesting key roles in this microbiome, with the identified dimethylsulfoniopropionate (DMSP) metabolisers having important functions in these habitats. The majority of those identified are involved in nutrient cycling or involved in the production of compounds that promote host survival. Increasingly, blue carbon and nature-based solutions to climate change are heralded as viable mitigation steps to limit climate change, however, this is done with little to no consideration of the microbial communities that cycle sequestered carbon in these environments. Here, we examine the microbial communities present in sediment samples taken in close proximity to each tree, sediment samples represent a major sink of atmospheric carbon and understanding how the associated communities will change as climate change advances will become an increasingly important part of carbon stock assessments. Knowing what microbes are presently there is an important first step in this process.

Distributed across both the tropical Atlantic and Pacific oceans, the seagrass Halophilia ovalis stabilizes coastal sediment, thereby preventing shoreline erosion and is also an important food source for megaherbivores such as dugongs. However, seagrass meadows globally are under severe duress due to both climate change and anthropogenic activities. We characterized the mycobiome of Halophilia ovalis at seven sites in the Malay Peninsula using ITS1 rDNA amplicon sequences and investigated differences in fungal community structure. We found that geographic location was a significant factor shaping fungal communities and that marine sediment harboured significantly higher diversity when compared to H. ovalis leaves, roots and rhizomes. Taken together, it is likely that locality rather than specific plant structure determines fungal community structure in H. ovalis. Because the plant mycobiome is known to exert a strong effect on plant health, to maximize the success of future seagrass transplantation and restoration work we propose that these efforts consider the importance of seagrass mycobiomes at all stages.

Though much is known about fungal importance to forest health, there is very little information about factors that impact soil fungal community recovery time after a fire. Soil samples were taken from burn sites within one ecotype of temperate coniferous forest in Utah over a 20-year chronosequence. Sites were selected from available historic burns and were similar in plant community structure, elevation, slope, and aspect. Fungal DNA from these samples was compared to soil from paired unburned sites nearby to measure community similarity and estimate soil fungal recovery rates. Differences between paired burned and unburned sites remained fairly stable over a decadal time scale overall, but fungal community structure was found to recover more quickly in areas with a higher average annual temperatures. A significant positive correlation in community recovery was seen in areas with a difference of as little as two degrees celsius per year. The only other environmental variable that significantly interacted with time since burn was annual mean precipitation. As global temperatures increase, alpine fires are increasing as well, but these results suggest that fungal community recovery time will be shortened under new climate scenarios.

Corals harbour diverse microbial communities that can change in composition as the host grows in age and size. Larger and older colonies have been shown to host a higher diversity of microbial taxa and this has been suggested to be a consequence of their more numerous, complex and varied micro-niches available. However, the effects of host age on community structure and diversity of microbial associates remain equivocal in the few studies performed to date. To test this relationship more robustly, we use established techniques to accurately determine coral host age by quantifying annual skeletal banding patterns, and utilise high-throughput sequencing to comprehensively characterise the microbiome of the common reef-building coral, Porites lutea. Our results indicate no clear link between coral age and microbial diversity or richness. Different sites display distinct age-dependent diversity patterns, with more anthropogenically impacted reefs appearing to show a winnowing of microbial diversity with host age, possibly a consequence of corals adapting to degraded environments. Less impacted sites do not show a signature of winnowing, and we observe increases in microbial richness and diversity as the host ages. Furthermore, we demonstrate that corals of a similar age from the same reef can show very different microbial richness and diversity.

A phylogenetically diverse array of fungi live within healthy leaf tissue of dicotyledonous plants. Many studies have examined these endophytes within a single plant species and/or at small spatial scales, but landscape-scale variables that determine their community composition are not well understood, either across geographic space, across climatic conditions, or in the context of host plant phylogeny. Here, we evaluate the contributions of these variables to endophyte beta diversity using a survey of foliar endophytic fungi in native Hawaiian dicots sampled across the Hawaiian archipelago. We used Illumina technology to sequence fungal ITS1 amplicons to characterize foliar endophyte communities across five islands and 80 host plant genera. We found that communities of foliar endophytic fungi showed strong geographic structuring between distances of 7 and 36 km. Endophyte community structure was most strongly associated with host plant phylogeny and evapotranspiration, and was also significantly associated with NDVI, elevation and solar radiation. Additionally, our bipartite network analysis revealed that the five islands we sampled each harboured significantly specialized endophyte communities. These results demonstrate how the interaction of factors at large and small spatial and phylogenetic scales shapes fungal symbiont communities.

SummaryFungi play pivotal roles in ecosystem functioning, but little is known about their global patterns of diversity, endemicity, vulnerability to global change drivers and conservation priority areas. We applied the high-resolution PacBio sequencing technique to identify fungi based on a long DNA marker that revealed a high proportion of hitherto unknown fungal taxa. We used a Global Soil Mycobiome consortium dataset to test relative performance of various sequencing depth standardization methods (calculation of residuals, exclusion of singletons, traditional and SRS rarefaction, use of Shannon index of diversity) to find optimal protocols for statistical analyses. Altogether, we used six global surveys to infer these patterns for soil-inhabiting fungi and their functional groups. We found that residuals of log-transformed richness (including singletons) against log-transformed sequencing depth yields significantly better model estimates compared with most other standardization methods. With respect to global patterns, fungal functional groups differed in the patterns of diversity, endemicity and vulnerability to main global change predictors. Unlike α-diversity, endemicity and global-change vulnerability of fungi and most functional groups were greatest in the tropics. Fungi are vulnerable mostly to drought, heat, and land cover change. Fungal conservation areas of highest priority include wetlands and moist tropical ecosystems.

15 páginas.- 6 figuras.- 93 referencias, Fungi are highly diverse organisms, which provide multiple ecosystem services. However, compared with charismatic animals and plants, the distribution patterns and conservation needs of fungi have been little explored. Here, we examined endemicity patterns, global change vulnerability and conservation priority areas for functional groups of soil fungi based on six global surveys using a high-resolution, long-read metabarcoding approach. We found that the endemicity of all fungi and most functional groups peaks in tropical habitats, including Amazonia, Yucatan, West-Central Africa, Sri Lanka, and New Caledonia, with a negligible island effect compared with plants and animals. We also found that fungi are predominantly vulnerable to drought, heat and land-cover change, particularly in dry tropical regions with high human population density. Fungal conservation areas of highest priority include herbaceous wetlands, tropical forests, and woodlands. We stress that more attention should be focused on the conservation of fungi, especially root symbiotic arbuscular mycorrhizal and ectomycorrhizal fungi in tropical regions as well as unicellular early-diverging groups and macrofungi in general. Given the low overlap between the endemicity of fungi and macroorganisms, but high conservation needs in both groups, detailed analyses on distribution and conservation requirements are warranted for other microorganisms and soil organisms., The bulk of the funding is derived from the Estonian Science Foundation (grants PRG632, PRG1170, PRG1615, MOBTP198), EEA Financial Mechanism Baltic Research Programme (EMP442), and Novo Nordisk Fonden (NNF20OC0059948).

Significance Although fungi in the air make up a disproportionately large part of particulate matter and affect both local and global biogeochemical cycles, the factors influencing their composition and diversity are less clear, especially over relatively long timescales. Here we leverage the longest time series of aerobiota to date from a remote sampling location on Hawaii Island to examine changes in fungi over time and in response to environmental conditions. Our results provide evidence for a relatively unstable fungal aerobiota, whose composition is likely influenced by the ability to disperse into the system, rather than season or climate. Our finding of core taxa throughout the sampling period indicates resistance among some fungi to natural environmental fluctuations and human-associated global change., Fungi are ubiquitous and often abundant components of virtually all ecosystems on Earth, serving a diversity of functions. While there is clear evidence that fungal-mediated processes can influence environmental conditions, and in turn select for specific fungi, it is less clear how fungi respond to environmental fluxes over relatively long time frames. Here we set out to examine changes in airborne fungi collected over the course of 13 y, which is the longest sampling time to date. Air filter samples were collected from the Mauna Loa Observatory (MLO) on Hawaii Island, and analyzed using Illumina amplicon sequencing. As a study site, MLO is unique because of its geographic isolation and high elevation, making it an ideal place to capture global trends in climate and aerobiota. We found that the fungal aerobiota sampled at MLO had high species turnover, but compositional similarity did not decrease as a function of time between samples. We attribute these patterns to neutral processes such as idiosyncratic dispersal timing and trajectories. Furthermore, the composition of fungi at any given point was not significantly influenced by any local or global environmental variables we examined. This, and our additional finding of a core set of persistent fungi during our entire sampling period, indicates some degree of stability among fungi in the face of natural environmental fluctuations and human-associated global change. We conclude that the movement of fungi through the atmosphere is a relatively stochastic process.

Marine fungal biodiversity remains vastly understudied, and even less is known of their biogeography and the processes responsible for driving these distributions in marine environments. We investigated the fungal communities associated with the seagrass Enhalus acoroides collected from Singapore and Peninsular Malaysia to test the hypothesis that fungal communities are homogeneous throughout the study area. Seagrass samples were separated into different structures (leaves, roots, and rhizomes), and a sediment sample was collected next to each plant. Amplicon sequencing of the fungal internal transcribed spacer 1 and subsequent analysis revealed significant differences in fungal communities collected from different locations and different structures. We show a significant pattern of distance decay, with samples collected close to each other having more similar fungal communities in comparison with those that are more distant, indicating dispersal limitations and/or differences in habitat type are contributing to the observed biogeographic patterns. These results add to our understanding of the seagrass ecosystem in an understudied region of the world that is also the global epicenter of seagrass diversity. This work has implications for seagrass management and conservation initiatives, and we recommend that fungal community composition be a consideration for any seagrass transplant or restoration programme., Examining marine fungi associated with the seagrass, Enhalus acoroides.

Marine environments abound with opportunities to discover new species of fungi even in relatively well-studied ecosystems such as coral reefs. Here, we investigated the fungal communities associated with the canopy forming macroalga Sargassum ilicifolium(Turner) C. Argardh (1820) in Singapore. We collected eight S. ilicifolium thalli from each of eight island locations and separated them into three structures—leaves, holdfast and vesicles. Amplicon sequencing of the fungal internal transcribed spacer 1 (ITS1) and subsequent analyses revealed weak but significant differences in fungal community composition from different structures. Fungal communities were also significantly different among sampling localities, even over relatively small spatial scales (≤ 12 km). Unsurprisingly, all structures from all localities were dominated by unclassified fungi. Our findings demonstrate the potential of marine environments to act as reservoirs of undocumented biodiversity that harbour many novel fungal taxa. These unclassified fungi highlight the need to look beyond terrestrial ecosystems in well-studied regions of the world, and to fully characterize fungal biodiversity in hotspots such as Southeast Asia for better understanding the roles they play in promoting and maintaining life on our planet.

The coral holobiont contains a diverse community of bacteria that have been widely acknowledged as a major contributor in the maintenance of host health and in promoting reef resilience under changing environments. However, little is known regarding the spatial distribution of these communities or the processes and mechanisms that are responsible for creating these patterns. Here we show that bacterial communities associated with the reef-building coral, Pocillopora acuta, from nine offshore islands in an urbanised coral reef ecosystem (Singapore) can diverge sharply and are significantly different among sampling locations. We suggest that small-scale environmental factors such as prevailing surface currents and wind direction, even over short distances (

Fungal endophyte (FE) communities can be shaped by environmental conditions and/or host-plant affinities. Hawaiʻi's landscape-dominant woody genus Metrosideros (Myrtaceae) comprises several vegetatively distinct taxa that are non-randomly distributed across environments, and their FE communities are poorly known. We examined the relative importance of Metrosideros taxon and environment (elevation) on FE communities on Oʻahu. ITS1 rDNA barcoding and sequencing of 113 trees detected a richness of 1,637 FEs representing 5 phyla, 223 genera, and 200 species. Variation in FE diversity was significantly explained by host taxon and site with considerable overlap in FE communities among taxa. FE communities did not vary between pubescent and glabrous taxa or across elevations, possibly due to the relatively narrow range of environmental conditions represented on Oʻahu relative to taller islands. A significant pattern of isolation by distance in FE composition was detected both among and within sites, consistent with restricted dispersal of FEs across the island.

Beneficial microbes such as plant mutualistic fungi, hold the promise of ameliorating challenges faced in native plant conservation such as disease management. As an alternative to costly chemical pest control, conservation efforts could potentially harness the benefits of plant mutualistic fungi to aid in defense and disease resistance, but there are few tests of this notion. We set out to test the efficacy of controlling a common foliar pathogen, the powdery mildew Neoerysiphe galeopsidis, by inoculating the endangered Hawaiian plant species Phyllostegia kaalaensis with potentially beneficial members of its wild-type mycobiome. We tested whether inoculating plants with above or belowground fungal mutualists, or both, led to increased disease resistance in the host. We found that while all treatments reduced average disease incidence, colonization by the foliar yeast Moesziomyces aphidis was the only treatment to do so significantly. These results provide an exciting new strategy for plant conservation practices.

A phylogenetically diverse array of fungi live within healthy leaf tissue of dicotyledonous plants. Many studies have examined these endophytes within a single plant species and/or at small spatial scales, but landscape-scale variables that determine their community composition are not well understood, either across geographic space, across climatic conditions, or in the context of host plant phylogeny. Here, we evaluate the contributions of these variables to endophyte beta diversity using a survey of foliar endophytic fungi in native Hawaiian dicots sampled across the Hawaiian archipelago. We used Illumina technology to sequence fungal ITS1 amplicons to characterize foliar endophyte communities across five islands and 80 host plant genera. We found that communities of foliar endophytic fungi showed strong geographic structuring between distances of seven and 36 km. Endophyte community structure was most strongly associated with host plant phylogeny and evapotranspiration, and was also significantly associated with NDVI, elevation, and solar radiation. Additionally, our bipartite network analysis revealed that the five islands we sampled each harbored significantly specialized endophyte communities. These results demonstrate how the interaction of factors at large and small spatial and phylogenetic scales shape fungal symbiont communities.

Microbes influence plant phenotypes but most known examples of this are from the study of below-ground microbes and plant disease modification. To examine the potential importance of phyllosphere microbes on non-disease related plant traits, we used sterile Arabidopsis clones to test the effects of foliar fungi on flowering phenology and reproductive allocation under conditions of varying water stress. We inoculated the sterile plants with fully-factorial combinations of four fungal isolates, then measured flowering time and reproductive allocation for each treatment group under normal and water-stressed conditions. All plants inoculated with foliar fungi had significantly later flowering and greater seed mass than the sterile control groups. The magnitude of this effect depended on the specific fungi present, but individual fungal effects diminished as inoculum richness increased. Above-ground microbes likely influence other plant traits as well and should be considered in any study measuring plant phenotypes.

Fundamental questions remain about the role of bacterivorous protists in regulating soil carbon (C) and nitrogen (N) cycling, including the ways in which protists interact with physical and chemical factors to influence soil decomposer responses to increased temperature. Amoebae in particular deserve attention given their status as one of the most abundant soil protozoans. Using microcosms of simplified soil communities, we investigated the net and interactive effects of amoebal bacterivory, soil aggregate structure, agricultural management (till vs. no-till), and temperature on C and N dynamics during a 24 day-incubation. The respiration temperature sensitivity (Q10) was much more variable for the simplified communities than natural communities, illustrating the challenge in using low-diversity systems to predict such a complex and emergent property. In addition to the expected positive effect of amoebal predation on C and N mineralization in all treatments, we found that the magnitude of this effect was significantly influenced by aggregate structure and temperature. Statistically higher (P

There has been very little effort to incorporate foliar microbiomes into plant conservation efforts even though foliar endophytes are critically important to the fitness and function of hosts. Many critically endangered plants that have been extirpated from the wild are dependent on regular fungicidal applications in greenhouses that cannot be maintained for remote out-planted populations, which quickly perish. These fungicides negatively impact potentially beneficial fungal symbionts, which may reduce plant defenses to pathogens once fungicide treatments are stopped. Using the host/parasite system of Phyllostegia kaalaensis and Neoerysiphe galeopsidis, we conducted experiments to test total foliar microbiome transplants from healthy wild relatives onto fungicide-dependent endangered plants in an attempt to mitigate disease and reduce dependency on fungicides. Plants were treated with total microbiome transplants or cultured subsets of this community and monitored for disease severity. High-throughput DNA screening of fungal ITS1 rDNA was used to track the leaf-associated fungal communities and evaluate the effectiveness of transplantation methods. Individuals receiving traditionally isolated fungal treatments showed no improvement, but those receiving applications of a simple leaf slurry containing an uncultured fungal community showed significant disease reduction, to which we partially attribute an increase in the mycoparasitic Pseudozyma aphidis. These results were replicated in two independent experimental rounds. Treated plants have since been moved to a native habitat and, as of this writing, remain disease-free. Our results demonstrate the effectiveness of a simple low-tech method for transferring beneficial microbes from healthy wild plants to greenhouse-raised plants with reduced symbiotic microbiota. This technique was effective at reducing disease, and in conferring increased survival to an out-planted population of critically endangered plants. It was not effective in a closely related plant. Plant conservation efforts should strive to include foliar microbes as part of comprehensive management plans.

Throughout the world DNA banks are used as storage repositories for genetic diversity of organisms ranging from plants to insects to mammals. Designed to preserve the genetic information for organisms of interest, these banks also indirectly preserve organisms’ associated microbiomes, including fungi associated with plant tissues. Studies of fungal biodiversity lag far behind those of macroorganisms, such as plants, and estimates of global fungal richness are still widely debated. Utilizing previously collected specimens to study patterns of fungal diversity could significantly increase our understanding of overall patterns of biodiversity from snapshots in time. Here, we investigated the fungi inhabiting the phylloplane among species of the endemic Hawaiian plant genus, Clermontia (Campanulaceae). Utilizing next generation DNA amplicon sequencing, we uncovered approximately 1,780 fungal operational taxonomic units from just 20 DNA bank samples collected throughout the main Hawaiian Islands. Using these historical samples, we tested the macroecological pattern of decreasing community similarity with decreasing geographic proximity. We found a significant distance decay pattern among Clermontia associated fungal communities. This study provides the first insights into elucidating patterns of microbial diversity through the use of DNA bank repository samples.

Mesophotic coral ecosystems are an almost entirely unexplored and undocumented environment that likely contains vast reservoirs of undescribed biodiversity. Twenty-four macroalgae samples, representing four genera, were collected from a Hawaiian mesophotic reef at water depths between 65 and 86 m in the ‘Au‘au Channel, Maui, Hawai‘i. Algal tissues were surveyed for the presence and diversity of fungi by sequencing the ITS1 gene using Illumina technology. Fungi from these algae were then compared to previous fungal surveys conducted in Hawaiian terrestrial ecosystems. Twenty-seven percent of the OTUs present on the mesophotic coral ecosystem samples were shared between the marine and terrestrial environment. Subsequent analyses indicated that host species of algae significantly differentiate fungal community composition. This work demonstrates yet another understudied habitat with a moderate diversity of fungi that should be considered when estimating global fungal diversity.

During the period of March 2004 to December 2007, samples of aerial litter (dead but still attached plant parts) and ground litter (dead plant material on the ground) were collected from 81 study sites representing a wide range of latitudes (34°S to 50°S) and a variety of different types of habitats throughout New Zealand (including Stewart Island and the Auckland Islands). The objective was to survey the assemblages of protosteloid amoebae present in this region of the world. Twenty-nine described species of protosteloid amoebae were recorded by making morphological identifications of protosteloid amoebae fruiting bodies on cultured substrates. Of the species observed, Protostelium mycophaga was by far the most abundant and was found in more than half of all samples. Most species were found in fewer than 10% of the samples collected. Seven abundant or common species were found to display significantly increased likelihood for detection in aerial litter or ground litter microhabitats. There was some evidence of a general correlation between environmental factors - annual precipitation, elevation, and distance from the equator (latitude) - and the abundance and richness of protosteloid amoebae. An increase in each of these three factors correlated with a decrease in both abundance and richness. This study provides a thorough survey of the protosteloid amoebae present in New Zealand and adds to a growing body of evidence which suggests several correlations between their broad distributional patterns and environmental factors.

The eisosome protein Pil1 is known to be implicated in the endocytosis of Ste3, but the precise biological function of it during endocytosis is poorly understood. Here, we present data to reveal Pil1's role in receptor-mediated endocytosis. Using live cell imaging, we show that endocytic patches carrying Abp1 and Las17 persisted much longer in PIL1-deficient cells. The loss of Pil1 also greatly affected both the scission efficiency and the frequency of formation of endocytic sites carrying Rvs161- and Rvs167-GFP. Furthermore, the mistargeting of the synaptojanins, Sjl1 and Sjl2, to the cytoplasm in pil1Δ cells suggests that Pil1 is required for the proper recruitment of the synaptojanins to endocytic sites. A severe motility defect of Abp1-GFP during its internalization in a codeletant of PIL1 and SJL2 indicates a functional interplay between them in endocytosis. Together, these results establish that Pil1 is involved in the recruitment of endocytic proteins to optimize endocytosis.